Functional Role of Pseudouridine on Zika Virus Gene Expression

假尿苷对寨卡病毒基因表达的功能作用

• 批准号: 10732617
• 负责人: Cara Theresia Pager
• 金额: $ 22.8万
• 依托单位: STATE UNIVERSITY OF NEW YORK AT ALBANY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-06-09 至 2025-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10732617
• 关键词: Affect; Affinity; Americas; Antisense Oligonucleotides; Antiviral Agents; Appearance; Biological Assay; Biotinylation; Cell Line; Cell Nucleus; Cells; Chemicals; Culicidae; Cytoplasm; Defect; Detection; Development; Disease Outbreaks; Enzymes; Flavivirus; Gene Expression; Genome; Goals; HIV-1; Health; Hepatitis C virus; Human; Immune response; Infection; Knowledge; Label; Length; Life Cycle Stages; Luciferases; Mass Spectrum Analysis; Methodology; Microcephaly; Modification; Molecular; Molecular Biology; Mutagenesis; Neurologic; Nuclear Export; Nucleotides; Plaque Assay; Polyribosomes; Positioning Attribute; Proteins; Pseudouridine; RNA; RNA Folding; RNA Interference; RNA Stability; RNA Viruses; RNA-Protein Interaction; Regulation; Replicon; Reporting; Research; Reverse Transcription; Role; Site; Site-Directed Mutagenesis; Stress; Structure; System; Techniques; Technology; Testing; Therapeutic; Time; Transcript; Translations; Untranslated Regions; Viral; Viral Genes; Viral Physiology; Viral Proteins; Virion; Virus; Virus Assembly; Virus Diseases; Western Blotting; Woman; ZIKA; ZIKV infection; Zika Virus; combinatorial; epitranscriptomics; hepatoma cell; improved; infancy; insight; mosquito-borne; new therapeutic target; next generation sequencing; novel; overexpression; therapeutic target; transcriptome sequencing; viral RNA; viral transmission

Zika virus (ZIKV) is a re-emerging mosquito-borne flavivirus, that during the 2015-2016 outbreak in the Americas was associated with severe and devastating developmental abnormalities, including microcephaly, in babies born from infected women. The single-stranded positive-sense RNA genome of ZIKV directs translation of viral proteins, functions as a template for replication of the genome and is packaged into new virus particles. These functions are in part regulated by specific RNA structures within the untranslated regions of the RNA, and via interactions with viral and cellular proteins. More recently RNA modifications have been shown to significantly affect the virus infectious cycle. Although more than 170 different RNA modifications are known to decorate natural RNAs, only N6-methyladenosine has been reported to affect ZIKV gene expression. The role of other RNA modifications on ZIKV RNA is presently unknown. Using biotinylated antisense oligonucleotides to affinity isolate virus RNA from cells and virions, and mass spectrometry to identify RNA modifications, we discovered that more than 30 different RNA modifications are present on ZIKV RNA. In this application we focus on pseudouridine (Y), an abundant but understudied RNA modification. The incorporation of Y on cellular RNAs affects the folding, stability, and translation of RNA, and RNA-RNA and RNA-protein interactions. We propose that Y on the ZIKV RNA genome promotes virus translation and replication. In this application we propose using a next generation sequencing approach combined with chemical modification of pseudouridine to identify specific Y sites on ZIKV genome (Aim 1). Next, we will undertake RNAi depletion and overexpression of cellular pseudouridine synthase proteins to identify the host enzymes responsible for installing pseudouridine on ZIKV RNA (Aim 2). Last, we will use mutagenesis and modulation of the pseudouridine synthase enzymes to elucidate if pseudouridine regulates ZIKV translation and/or replication (Aim 3). Moreover, these studies will be undertaken in the context of virus infection in mammalian and mosquito cells, two hosts biologically relevant to the virus life cycle, which could inform the role of RNA modifications in virus transmission and host adaptation. We expect that this research will advance our knowledge of RNA modifications which have been shown to be novel and important regulators of viral gene expression and improve our understanding of the molecular biology of ZIKV. This research will therefore provide critical information on a virus with significant impacts on human health and that currently lacks therapeutic options.

寨卡病毒 (ZIKV) 是一种重新出现的蚊媒黄病毒，该病毒在 2015 年至 2016 年美洲爆发期间与受感染妇女所生婴儿的严重和毁灭性发育异常有关，包括小头畸形。 ZIKV 的单链正义 RNA 基因组指导病毒蛋白的翻译，充当基因组复制的模板，并被包装成新的病毒颗粒。这些功能部分受到 RNA 非翻译区域内特定 RNA 结构的调节，并通过与病毒和细胞蛋白质的相互作用来调节。最近，RNA 修饰已被证明可以显着影响病毒的感染周期。尽管已知超过 170 种不同的 RNA 修饰可修饰天然 RNA，但据报道，只有 N6-甲基腺苷会影响 ZIKV 基因表达。其他 RNA 修饰对 ZIKV RNA 的作用目前尚不清楚。使用生物素化反义寡核苷酸从细胞和病毒体中亲和分离病毒 RNA，并通过质谱法鉴定 RNA 修饰，我们发现 ZIKV RNA 上存在 30 多种不同的 RNA 修饰。在此应用中，我们重点关注假尿苷 (Y)，这是一种丰富但尚未得到充分研究的 RNA 修饰。 Y 在细胞 RNA 上的掺入会影响 RNA 的折叠、稳定性和翻译，以及 RNA-RNA 和 RNA-蛋白质相互作用。我们认为 ZIKV RNA 基因组上的 Y 促进病毒翻译和复制。在此应用中，我们建议使用下一代测序方法结合假尿苷的化学修饰来识别 ZIKV 基因组上的特定 Y 位点（目标 1）。接下来，我们将进行 RNAi 耗竭和细胞假尿苷合酶蛋白的过度表达，以确定负责在 ZIKV RNA 上安装假尿苷的宿主酶（目标 2）。最后，我们将利用假尿苷合酶的诱变和调节来阐明假尿苷是否调节 ZIKV 翻译和/或复制（目标 3）。此外，这些研究将在哺乳动物和蚊子细胞中的病毒感染背景下进行，这两种宿主在生物学上与病毒生命周期相关，这可以了解RNA修饰在病毒传播和宿主适应中的作用。我们期望这项研究将增进我们对 RNA 修饰的认识，RNA 修饰已被证明是病毒基因表达的新颖且重要的调节因子，并提高我们对 ZIKV 分子生物学的理解。因此，这项研究将提供有关对人类健康产生重大影响且目前缺乏治疗选择的病毒的重要信息。